The present invention relates to methods for purifying sugars in particular methods for desalting oligosaccharides and their derivatives.
In contrast to proteins which, because of their high molecular weight, are readily desalted by dialysis or gel chromatography, small oligosaccharides are extremely difficult to desalt. Methods which are used include the removal of ions using mixed-bed ion-exchange columns, precipitation of the salt by a non-aqueous solvent or chemical reaction (such as adding barium carbonate to remove sulfuric acid as the insoluble barium sulfate) and by gel chromatography using highly-cross-linked packings (with which separation of small oligosaccharides is difficult to achieve).
Solid-phase extraction cartridges are now commercially available for the extraction of hydrophobic substances from aqueous solution, such as pesticide and hydrocarbon residues in ground water. Most commonly, these contain silica which has been modified by the covalent incorporation of allyl groups (most commonly octadecyl or ODS chains) to confer reversed-phase separation characteristics. Such reversed-phase adsorbents show excellent retention of hydrophobic substances, but have very little affinity for hydrophilic solutes, such as sugars. These cartridges are therefore useful for the removal of salts from hydrophobic substances, but not from sugars.
Activated carbon is a long-established adsorbent for the purification of both gases and liquids. It has also been applied to the preparative chromatographic fractionation of mixtures of oligosaccharides which are obtained by partial hydrolysis of polysaccharides, such as starch (1). In that application, an inert diluent, typically diatomaceous earth, is mixed with the carbon to enable increased flow of the solvent through the column. A solution of the crude hydrolysate is applied to such a xe2x80x98charcoal-Celitexe2x80x99 column and the different oligosaccharides are obtained, in order of increasing size. by elution with water containing increasing proportions of ethanol as an organic modifier. The composition is typically changed on a batch basis, rather than by elution with a gradient of organic modifier.
Activated carbon chromatographic adsorbents have not been used in high-performance liquid chromatography (HPLC) but, with the recent availability of graphitised carbon packings (2) an analogous separation mode has become available. Like the older xe2x80x98activated carbonxe2x80x99 and xe2x80x98activated charcoalxe2x80x99, graphitised carbon has extremely high chemical stability, as well as good mechanical properties.
Porous graphitised carbon is presently manufactured by Shandon HPLC (Cheshire, England) who also markets Hypercarb HPLC columns. Carbograph solid-phase extraction cartridges containing the same packing are available from Alltech Associates Inc (Deerfield, Ill. USA). HPLC separations of oligosaccharides have been reported using the Hypercarb column (3, 4, 5,6).
A hitherto unrecognised aspect is the value of carbon columns for the desalting of oligosaccharide solutions. This fact is surprising, in view of the lack of effective methods for the desalting of oligosaccharides. The present inventors have developed a useful method to remove salts or polypeptides from solutions of oligosaccharides.
In a first aspect, the present invention consists in a method to separate oligosaccharides from salts comprising the steps of:
(a) reacting an oligosaccharide solution including salts with a solid support comprising carbon such that the oligosaccharides substantially bind to the solid support;
(b) washing the support to remove salts; and
(c) eluting the bound oligosaccharides from the support.
In a second aspect, the present invention consists in a method to separate oligosaccharides from polypeptides comprising the steps of:
(a) reacting an oligosaccharide solution including polypeptides with a solid support comprising carbon such that the oligosaccharides substantially bind to the solid support;
(b) washing the support to remove polypeptides; and
(c) eluting the bound oligosaccharides from the support.
Preferably, the solid support comprises porous graphitised carbon and the support is packed in a chromatography column or cartridge. In these forms the support can be used in either continuous or batch mode, for clean-up of samples before chromatography, for chromatographic separation or for clean-up after chromatography, especially when samples are to be submitted to spectroscopic analysis. The washing step is preferably with water and the elution step preferably includes an organic modifier. In one preferred form, the organic modifier is acetonitrile. The concentration of organic modifier is preferably from about 1 to 90% v/v. It will be appreciated by one skilled in the art, however, that other organic modifiers are suitable, especially the lower alcohols methanol, ethanol, propanol and butanol.
The bound oligosaccharides can be removed by varying the concentration of the organic modifier during the elution step. Furthermore, in order to separate different oligosaccharides bound to the support, different concentrations of organic modifier can be used in combination with acidic or basic modifiers. A suitable acidic modifier is a dilute acid, for example, trifluoroacetic acid (TFA), preferably at a concentration of about 0.01 to 1% v/v. A suitable basic modifier is ammonia. It will be appreciated, however, that other basic and acidic modifiers would be suitable for the methods of the present invention. The concentration of acidic or basic modifiers is preferably from about 0.01 to 5% v/v. Elution may include increasing the concentration of organic modifier in steps or by gradient. Such elution techniques are well known to one skilled in the art.
The methods of the present invention are particularly useful in removing salts used in biological buffers and reagents, as well as removing polypeptides from sugar solutions. Examples include the salts sodium chloride and potassium chloride, caustic alkali like sodium hydroxide, cationic buffers like Tris, anionic buffers like phosphate and acetate, zwitterionic buffers like HEPES and MOPS, and surfactants like SDS. It will be appreciated by one skilled in the art that the salts listed above are only provided as examples and many other salts can be removed from oligosaccharides using the method of the present invention. Furthermore, other compounds used in the separation and analysis of biological materials that do not fall within the strict chemical definition of xe2x80x9csaltsxe2x80x9d may also be removed from sugars using the methods of the present invention. Examples of such compounds are non-ionic detergents. Removal of these compounds from sugars is included within the scope of the present invention.
In order that the present invention may be more clearly understood, preferred forms thereof will be described with reference to the following drawings: